Staphylococcus aureus is a Gram-positive human pathogen that causes a wide range of infections from skin and soft tissue infections (SSTI) to life threatening sepsis and pneumonia. The pathogenicity of S. aureus is dependent on numerous virulence factors, including cell surface proteins and polysaccharides, as well as secreted toxins. An important group of these toxins includes pore forming toxins such as hemolysins and leukocidins, as well as small peptidic toxins, with lytic activity towards erythrocytes and leukocytes particularly the neutrophils that represent the major line of defense against S. aureus. These toxins cause tissue damage, promote bacterial dissemination and growth in distant organs, and enable the pathogen to evade the host innate immune response. Furthermore, capsular polysaccharide (CP) are known to protect bacteria from phagocytic activity of polymorphonuclear leukocytes and have been validated as important vaccine targets. Integrated BioTherapeutics (IBT) is pursuing development of toxin- based vaccines in combination with CPs against S. aureus infections. The pore-forming ?-hemolysin (Hla), also known as ?-toxin (AT), is produced by nearly all strains and is implicated in several S. aureus invasive diseases. Recent microbiological and serological studies in humans with S. aureus bacteremia also show the importance of ?-toxin and phenol-soluble modulins (PSM) for S. aureus pathogenicity. Based on our recent study, pre-existing antibodies against Hla, ?-toxin and PSM?3 significantly reduce the risk of sepsis in adults with S. aureus bacteremia. The goal of this Phase I SBIR is to evaluate the feasibility of a novel glycoconjugate staphylococcal toxin based vaccine. Vaccine candidates have been designed that represent a critical structural domain at the N terminus of Hla (AT62) fused to ?-toxin and PSM?3. We have shown previously that AT62 vaccine provides protection against bacteremia and pneumonia. This proposal utilizes a unique and novel in vivo bioconjugation technology developed by Glycovaxyn Inc. to conjugate this hybrid toxoid with capsular polysaccharide Type 5 (CP5) as proof of concept of the efficacy of the bioconjugate. The bioconjugation technology involves E. coli cells engineered to express the Campylobacter enzyme PglB as well as the genes required for S. aureus CP8 biosynthesis. PglB transfers N-linked CP8 chains to the vaccine construct introduced into these cells. This novel technology is a major development in glycoconjugate vaccine field with multiple advantages over the conventional chemical conjugation. In the Specific Aim 1 fusions of the three toxins will be generated and attenuating mutations identified and incorporated into the fusion construct. The candidate vaccine will be tested for immunogenicity and then bioconjugates generated in Aim 2. In Aim 3 the protective efficacy of the vaccine candidate will be evaluated in two major disease models representing bacteremia/sepsis and skin and soft tissue infections. Upon successful completion of this proposed research we envision a Phase II SBIR in which similar bioconjugate will be produced for the other major S. aureus capsular polysaccharide CP8 and the efficacy of the multivalent vaccine (including combination other toxoid vaccines currently under development in our group) extensively tested. The ultimate goal is to build a strategic partnership with large pharma to transition the vaccine candidate into clinical development.